Prasanthi Chengalva et al, J.

Global Trends Pharm Sci, 2017; 8(2): 4010 - 4015

An Elsevier Indexed Journal ISSN-2230-7346

Journal of Global Trends in Pharmaceutical Sciences

NOVEL ANALYTICAL METHOD FOR THE SIMULTANEOUS ASSAY OF

TIZANIDINE AND MEFENAMIC ACID BY RP-HPLC

Prasanthi Chengalva*1, Aruna Gundala2

1Department of Pharmaceutical Analysis, Krishna Teja Pharmacy College, Tirupathi-517501,
Chittoor (Dt), Andhra Pradesh, India.
2 Department of Pharmaceutical Analysis and Quality Assurance, Krishna Teja Pharmacy College,

Tirupathi-517501, Chittoor (Dt), Andhra Pradesh, India.

*Corresponding author E-mail: prashanthi.chengalva87@gmail.com

ARTICLE INFO ABSTRACT

A simple, economic, rapid, accurate and precise analytical
Key Words method for simultaneous determination of Tizanidine and Mefenamic
RPHPLC, Method acid in bulk and pharmaceutical dosage form was developed.
development, Chromatographic separation was carried out using Symmetry C18
validation, Tizanidine, (2504.6 mm5 m) with a mobile phase consisting of phosphate buffer
Mefenamic acid (pH 7.0) and acetonitrile in the ratio 55:45%v/v at a flow rate of 1
ml/min at a detection wavelength of 274 nm. The developed method
resulted in the elution of Tizanidine and Mefenamic acid at 3.407 min
and 4.750 min respectively. The calibration curves were linear
(r=0.999) in the concentration range of 1-3 g/ml for Tizanidine and
12.5-37.5 g/ml for Mefenamic acid. The % RSD for Tizanidine and
Mefenamic acid was found to be 1.55 and 0.64 respectively. The %
mean recovery was found to be 99.65-100.25% for Tizanidine and
99.57-100.31% for Mefenamic acid. The LOD was found to be 0.08
g/ml and 1.12 g/ml and LOQ was found to be 0.25 g/ml and 3.67
g/ml for Tizanidine and Mefenamic acid respectively. The proposed
HPLC method was found to be simple, specific, precise, accurate, rapid
and economical for simultaneous estimation of Tizanidine and
Mefenamic acid in drug substance and drug products.

INTRODUCTION:
treat pain during periods, heavy bleeding
Tizanidine is a central alpha-2-
during periods, fever and inflammation. A
adrenergic receptor agonist and presumably
detailed literature survey revealed that there
reduces spasticity by increasing presynaptic
were analytical methods for the estimation
inhibition of motor neurons. It is chemically
of Tizanidine and Mefenamic acid alone and
5-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-
with other combinations1-9. Ganshyam
2,1,3-benzothiadiazol-4-amine (fig. no. 1).
et.al.,(2017) and Ashok et.al.,(2009)
Mefenamic acid is an anthranilic acid
reported spectroscopic methods for the
derivative belongs to NSAIDs. It is
simultaneous estimation of Tizanidine and
chemically 2- [(2,3-dimethylphenyl)amino]
Mefenamic acid10,11 but no HPLC methods
benzoic acid (fig. no. 2). A combination of
reported for the simultaneous estimation of
Tizanidine and Mefenamic acid is used to
Tizanidine and Mefenamic acid. Hence an

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 Prasanthi Chengalva et al, J. Global Trends Pharm Sci, 2017; 8(2): 4010 - 4015

attempt has been made to develop a rapid, Chromatographic conditions

sensitive, accurate and precise RPHPLC
method for the simultaneous estimation of
Tizanidine and Mefenamic acid in bulk drug
and pharmaceutical formulation.
Figure 1. Structure of Tizanidine
The developed method used a
reverse phase Symmetry C18 column
(250x4.6 mm, 5m), a mobile phase of
phosphate buffer (pH 7.0): Acetonitrile
(55:45), flow rate of 1.0 ml/min and a
detection wavelength of 274 nm using a UV
detector.
Preparation of standard solution
Standard stock solution of
Tizanidine and Mefenamic acid were
prepared by dissolving 2 mg of Tizanidine
and 25 mg of Mefenamic acid in sufficient
mobile phase. After that, the solution was
sonicated for 5 min and diluted to 100 ml
with mobile phase. Further dilutions were
prepared in 5 replicates of 2 g/ml
Tizanidine and 25 g/ml Mefenamic acid
which was made by adding 1 ml of stock
solution to 10 ml of mobile phase.

Figure 2. Structure of Mefenamic acid

MATERIALS AND METHODS

Materials
HPLC grade Mono potassium
dihydrogen phosphate (KH2PO4),
Dipotassium hydrogen phosphate (K2HPO4)
and acetonitrile were purchased from Merck
(Mumbai, India). Pharmaceutical grade Figure 3. UV spectrum of Tizanidine
Tizanidine and Mefenamic acid were
supplied as a gift sample by Chandra labs
(Hyderabad, India) and Mef T tablets
(Tizanidine 2 mg & Mefenamic acid 25 mg)
were purchased from local pharmacy.
Methods
Selection of wavelength
Standard solutions of Tizanidine and
Mefenamic acid were prepared at the
concentration of 10 g/ml scanned by
UV/Vis spectrophotometer at the range of Figure 4. UV spectrum of Mefenamic
200-400 nm. UV spectrums of Tizanidine Acid
(fig. no. 3) and Mefenamic acid (fig. no. 4)
were shown below. The isobestic point Preparation of sample solution12
selected for simultaneous estimation was 20 tablets each containing 2 mg of
274 nm (fig. no. 5). Tizanidine and 25 mg of Mefenamic acid
were weighed and taken into a mortar and

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crushed to fine powder and uniformly
mixed. Sample solution of Tizanidine and
Mefenamic acid were prepared by dissolving
weight of tablet powder equivalent to 2 mg
of Tizanidine and 25 mg of Mefenamic acid
and dissolved in sufficient mobile phase.
Then the solution was sonicated for 5 min,
filtered and diluted to 100 ml with mobile
phase. Further dilution of 2 g/ml Tizanidine
and 25 g/ml Mefenamic acid was made by
adding 1 ml of stock solution to 10 ml of
mobile phase.
Preparation of buffer solution
61.5 ml of 1M KH2PO4 and 38.5 ml
of 1M K2HPO4 were taken into 200 ml of
water, m9xed well and volume was made up
to 1000 ml with water. The buffer was
filtered through 0.45 filter and sonicated
for 20 min. The pH of the resulted solution
was found to be 7.0.
done on a C18 column in isocratic HPLC
mode with a mobile phase consisting of
phosphate buffer (pH 7.0): Acetonitrile
(55:45). A typical RP-HPLC chromatogram
for simultaneous determination of
Tizanidine and Mefenamic acid was shown
in (fig. no. 6).
Method validation
The developed RP-HPLC method
was validated for parameters like system
suitability, linearity, accuracy, precision,
limit of detection (LOD), limit of
quantitation (LOQ) and robustness
according to ICH guidelines13.

Figure 6. Chromatogram of Tizanidine

and Mefenamic acid
System suitability
Standard solutions were prepared as
Figure 5. UV overlap spectrum of per the test method and injected into the
Tizanidine and Mefenamic acid chromatographic system. The system
hydrochloride suitability parameters like theoretical plates,
RESULTS AND DISCUSSION resolution and asymmetric factor were
evaluated. The system suitability parameters
Method development
were tabulated in (Table no. 1). All the
Different chromatographic parameters were found to be within the
conditions were tried for better separation limits.
and resolution. Symmetry C18 (2504.6mm,
5m) column was found satisfactory. Peak
purity of Tizanidine and Mefenamic acid
was checked using UV detector and 274 nm
was considered satisfactory for detecting
both the drugs with adequate sensitivity. A
number of solvents in the different ratio over
a wide range of pH were tried, but either
peak shape was broad or resolution was not
good. Repeated trials to obtain good, sharp
peak with an efficient resolution between Figure 7. Calibration curve of Tizanidine
two peaks of Tizanidine and Mefenamic acid

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Table 1. System suitability data for Tizanidine and Mefenamic acid

Retention Tailing
Analytes Resolution Theoretical plates
time factor
Tizanidine 3.407 min - 1.822 4250
Mefenamic acid 4.750 min 12.854 1.134 7695
Table 2. Linearity data of Tizanidine and Mefenamic acid
Level Tizanidine Mefenamic acid
(%) Concentration (g/ml) Peak area Concentration (g/ml) Peak area
50 1.0 372.27 12.50 1379.78
75 1.5 558.40 18.75 2069.67
100 2.0 744.54 25.00 2759.56
125 2.5 930.67 31.25 3449.45
150 3.0 1106.81 37.50 4099.35
Correlation coefficient 0.999 0.999
Table 3. Method precision data of Tizanidine and Mefenamic acid
Tizanidine Mefenamic acid
n
Area Area
Injection 1 743.60 2762.23
Injection 2 754.48 2716.63
Injection 3 744.86 2759.56
Injection 4 725.80 2737.84
Injection 5 756.01 2741.33
Injection 6 755.47 2759.76
Average 746.70 2746.23
SD 11.60 17.81
%RSD 1.55 0.64
Table 4. Accuracy data of Tizanidine and Mefenamic acid
Tizanidine Mefenamic acid
Level (%) % Recovery % Mean* % Recovery % Mean*
50 99.75 99.12
50 99.25 99.82 99.98 99.57
50 100.48 99.62
100 99.75 100.50
100 99.83 99.65 99.07 100.04
100 99.37 100.56
150 100.16 100.11
150 100.08 100.39
100.25 100.31
150 100.51 100.44

Linearity
Linearity was evaluated by analysis of
standard solutions of Tizanidine and
Mefenamic acid of five different
concentrations from 50-150 % of target
concentration. The range of linearity was
found 1-3 g/ml for Tizanidine and 12.5-
37.5 g/ml for Mefenamic acid. The peak
area ratio and concentration of each drug
Figure 8. Calibration curve of Mefenamic was subjected to regression analysis to
acid calculate the calibration equations and

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Table 5. Robustness data for Tizanidine and Mefenamic acid
Parameter Tizanidine
Retension
Tailing*
time*
Less flow rate (0.8 4.377 1.951
ml/min)
More flow rate (1.2 2.930 1.922
ml/min)
Less wavelength 3.507 1.971
(272nm)
More wavelength
3.530 1.974
(276 nm )
* mean of 6 observations
Correlation coefficients (fig. no. 7 and 8).
The regression data obtained for Tizanidine
and Mefenamic acid was represented in
(Table no. 2). The result shows that within
the concentration range mentioned above,
there was an excellent correlation between
peak area ratio and concentration.
Limit of Detection (LOD) and Limit of
Quantitation (LOQ)
The LOD was calculated using the
formula 3.3 times /s where is the standard
deviation of the intercept obtained for
calibration curve and s is the slope of the
calibration curve. The LOD of Tizanidine
and Mefenamic acid was found to be 0.08
g/ml and 1.12 g/ml respectively. Similarly
LOQ is calculated using the formula 10
times /s. The LOQ of Tizanidine and
Mefenamic acid was found to be 0.25 g/ml
and 3.67 g/ml respectively.
Precision: The method precision was
demonstrated by injecting standard solutions
of Tizanidine and Mefenamic acid as per the
test procedure and the chromatograms of six
standard solutions were recorded. The
results of precision were tabulated in (Table
3). The % RSD of Tizanidine and
Mefenamic acid was found to be 1.55 and
0.64 respectively. % RSD values were
within the limits and the method was found
to be precise.
Accuracy: Accuracy of the developed
method has been carried out by recovery
studies by applying the standard addition
method. A known quantity of standard drug
Mefenamic acid
Plate Retension Plate
Tailing*
count* time* count*
4255 5.960 1.136 7698
4245 3.997 1.133 7692
4253 4.790 1.133 7693
4254 4.773 1.138 7692
concentration corresponding to 50%, 100%,
and 150% were added to preanalysed sample
solutions. Each set of addition were repeated
three times. The accuracy was expressed as
the percentage of analytes recovered by the
assay. The (Table no. 4) lists the recoveries
of the drug from a series of spiked
concentrations. The results indicate the
method was found to be accurate.
Robustness:
To determine the robustness of the
developed method, experimental conditions
were deliberately altered, and the system
suitability parameters were evaluated. The
solutions were prepared as per the test
method and injected at different variable
conditions like flow rate ( 0.2 ml/min.) and
wavelength ( 2 nm), system suitability
parameters were compared. The results were
tabulated in (Table no. 5). At the flow rate of
1.0 ml/min and wave length of 274 nm,
shows a sharp peak with good resolution and
rest of the flow rates and wave lengths were
found to be not satisfactory but passed all
system suitability parameters indicating that
the method was robust.
CONCLUSION
The proposed RP-HPLC method was
found to be simple, rapid, sensitive, accurate
and precise. Hence the developed method
can be useful for routine analysis of
Tizanidine and Mefenamic acid in bulk and
pharmaceutical formulation.
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ACKNOWLEDGEMENT equation methods. Indian J Pharm

Sci 2008;70:108-11.
The author expresses sincere thanks
8. Shah D, Jainika P, Rana, Usmangani
to the Principal, Krishna Teja Pharmacy
K, Baldania SL, Bhatt KK.
College for providing facilities to carry out
Development and validation of a
the work.
Liquid Chromatographic method for
CONFLICT OF INTERESTS estimation of Dicyclomine
Declare none hydrochloride, Mefenamic acid and
Paracetamol in tablets. Indian J
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